19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2β2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(β) and Y731(α) across the α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.


Measurements of RNR activity
The specific activity SA of RNR proteins was measured by the spectrophotometric assay 1,2 using a Cary 100 UV-Vis spectrometer (Agilent) at 25 °C. The temperature was controlled by a circulating water flow bath.
The influence of glycerol on wt-RNR activity was measured as following. The final concentrations in the assay amounted to 0.75 µM for wt-2, 0.15 µM for wt-2, 80 µM E. Coli thioredoxin (TR), 0.5 µM, E. Coli thioredoxin reductase (TRR), 1 mM CDP, and 3 mM ATP in assay buffer (50 mM HEPES, 15 mM MgSO4, 1 mM EDTA, pH 7.6) and glycerol contents of 0, 9, 18 and 27 v%. Practically, the concentrations were established in the cuvette by mixing 200 µL of a mastermix containing all ingredients except wt-2 at twice the concentration in assay buffer with an equal volume of assay buffer premixed with adequate amounts of glycerol. The reaction was initiated by first adding a volume of NADPH solution (ca. 7 µL, 11.8 mM) and then, after measuring background NADPH consumption, 4 µL of 15 µM wt-2-RNR (1.2 Y122•/2). 1 For each glycerol content, at least three runs were performed. The results are summarized in Figure S1 and Table S1. The SA values agree with reported wt-activities 1,2 and indicate that the high glycerol contents used for EPR sample preparation do not preclude activity of RNR.
The procedure for measuring the SA of E52QF3Y122-2/F2Y731-2 and F3Y122-2/F2Y731-2 was similar to the procedure described above, the results are summarized in Table  S1. E52QF3Y122-2/F2Y731-2 solution shows no SA within error. F3Y122-2/F2Y731-2 on the other hand clearly shows SA amounting to 6.8 ± 0.1% of the wt-enzyme. This value is in the expected range, given that the activities of wt-2/F2Y731-2 and F3Y122-2/wt-2 have been reported as 60 ± 10% 3 and 20 ± 10%, 1,4,5 respectively. E52QF3Y122-2/F2Y731-2 0 0 ± 10 E52QF3Y122-2/ 2 1 5 6 b,c a The SA of the 2 subunit was assayed. b Assay conditions are detailed in the corresponding references. c The reported residual SA was potentially caused by traces of co-expressed wt-2. Figure S1. Relative NADPH consumption rates of wt-RNR turnover as a function of glycerol content, normalized to the highest observed rate (Table S1).

EPR sample preparation
The most important samples have been described in the main text. Here, we give information on additional samples. All samples are summarized in Table S2.
In addition to the W-band samples containing glycerol as described in the main text, also samples with faster freezing points (TQ =11 -13 s) have been prepared without glycerol. These samples could be used to record EPR spectra at W-band, but the phase memory time TM was found to be too short for sensitive ENDOR measurements.
We also tried to prepare samples in which the  subunit (along with effector and substrate) was premixed with glycerol to achieve considerably faster quenching. However, this procedure led to visible precipitation of protein after mixing with the  subunit, even though premixing with glycerol was possible under the conditions of the spectrophotometric assay.
For Q-band PELDOR measurements, samples with RNR subunit concentrations of only 40 µM have been prepared in an analogous fashion as the samples at ~100% higher concentration described in the main text. Furthermore, E52QF3Y122-2/wt-2 (150 µM, 20v% glycerol) was prepared for an additional reference measurement.
For performing W-band 17 O ENDOR control experiments, Y356• was trapped using either F3Y122-2 or E52QF3Y122-2 in combination with 17 O-Y-2 in an analogous fashion to the other samples in 0.5 mm ID suprasil tubes (concentrations and TQ values are listed in Table S2). Additional information on the high-frequency set-up is described in a recent publication. 12 Echo detected EPR spectra at 263 GHz are presented in Figure S2 A and B, where each spectrum contains contributions from F3Y122 • and Y356 • . The contribution of Y356 • to the spectra in Figure S2 varies due to different radical yields in the different samples. As a representative example, Figure S3 illustrates the decomposition of the spectrum of F3Y122-2/F2Y731-2 into its contributions, which is achieved by subtraction of a reference spectrum of F3Y122 • .

34 GHz (Q-band) EPR spectra
Q-band EPR spectra at 10 K allow quantifying the yield of trapped Y356• as demonstrated on one representative example in Figure S4. Y356• yields in the F3Y122-2/F2Y731-2 samples turned out lower (typically 15 -20 %) than in the E52Q samples (typically 30 -35 %). Both are within the typical range observed previously (20 -40 %). [6][7][8] It is noted, that the samples for Q-and W-band measurements were prepared as one batch, leading to basically identical radical yields for Q-and W-band samples. Figure S4. Representative example of a Q-band EPR spectrum of an F3Y122-2/F2Y731-2 sample recorded at 10 K (black lines) along with a comparison to F3Y122• and Y356• reference spectra (blue and red lines, respectively). The reference spectra were obtained as described earlier. 6

34 GHz (Q-band) measurements of the phase memory time TM
TM measurements of Y356• at Q-band and T = 50 K were performed by measuring the echo intensity as a function of the interpulse separation  at three observer positions O1, O2, and O3 marked in Figure S5, which were also used for subsequent PELDOR experiments. The echo intensity I as function of  could be fit satisfactorily using a biexponential decay function (Equation S1): The biexponential fitting function allows for contributions of two radical species, as F3Y122• is detectable in all samples at this temperature. The obtained fitting parameters are tabulated in Table S3. Notably, TM,2 was slightly higher for the F3Y122-2/F2Y731-2 samples, which allowed for longer observation windows (see Figure 4, main text).

34 GHz (Q-band) PELDOR
Since orientation selectivity is observed in PELDOR measurements on tyrosyl radicals in RNR, three time traces with different observer and pump positions ( Figure S5) were recorded and summed up to reduce the effects of orientation selectivity on the analysis of the time traces. An example for the resulting orientation selective PELDOR time traces is shown in Figure S5. The results of all PELDOR measurements are summarized in Table S4.

EPR measurements at 50 K
A representative echo detected EPR spectrum of an F3Y122-2/F2Y731-2 at 50 K with a short  value of 240 ns is displayed in Figure S8. Under these conditions as well as under the conditions used for measuring the ~1.6 MHz 19 F HFC, both F3Y122• and Y356• contribute to the EPR spectrum, as is also demonstrated in Figure S8. Under the conditions used for measuring the < 250 kHz 19 F HFCs, the contribution of F3Y122• is suppressed because of its faster relaxation compared to Y356•. Figure S8. Representative example of a W-band EPR spectrum of an F3Y122-2/F2Y731-2 sample recorded at 50 K with  = 240 ns (black lines) along with a comparison to spectrum of F3Y122• (red). The blue spectrum was obtained by subtraction (black minus red). A resonator background signal which amounted to < 10% of the total intensity was subtracted.

Relaxation measurements at 50 K
The spin lattice relaxation time T1 of Y356• was measured with the inversion recovery (IR) sequence. All samples used for 19 F ENDOR measurement yielded similar results. The IR curves were fitted using Eq. (S1), with two components in almost equal weights having time constants of 300 µs and 1650 µs. At 2000 µs, which is the value used as SRT throughout the ENDOR experiments for all samples, more than 85% of the signal is recovered.
For the phase memory time TM, echo decay curves using the three-pulse echo sequence with a variety of T values were performed. TM for each T value was estimated by fitting Equation (S1) to the resulting time traces. Table S5 summarizes representative results obtained on E52QF3Y122-2/F2Y731-2 at 50 K.
Finally, we also tested some of the samples which were prepared without addition of glycerol. These samples had notably lower phase memory times (typically 25 -30 % faster relaxation, data not shown), thereby yielding lower sensitivities despite having higher concentrations, which prohibited their use for ENDOR.

Couplings ≲250 kHz
The strategy for choosing  when measuring small couplings aimed at high resolution while simultaneously avoiding the occurrence of a disturbing proton background signal.
-dependence of Mims ENDOR sensitivity. For the small couplings, the same procedure as in Ref. 11 was applied, and the sensitivity S of the ENDOR experiment was optimized using Equation (S2) TM values on the order of 700 -900 ns (Table S5)

Background correction in 50 K 19 F ENDOR spectra
Two different background signals have to be considered in the 19 Figure  S9 shows the orientation selective ENDOR spectra of F3Y122-2/Y730F-2 (purple lines) and F3Y122-2 (green lines). In the case of F3Y122-2/Y730F-2, the most significant proton contribution is observed at the gz position. At the gx position, the 1 H resonances are weak and spread out over a larger area. For the 19 F background, the most significant contribution is observed at the gx position, whereas the gz position has essentially no background signal. Background subtraction. The smoothed spectra shown in Figure S9 were subtracted from the 19

EPR measurements at 80 K
W-band EPR and ENDOR measurements on Y356• were performed at 80 K. The results show that the signal of unreacted F3Y122• is largely suppressed even for short  values (~250 ns) owed to its fast relaxation. This is illustrated in Figure S12 by the echo detected EPR spectrum of F3Y122-2/F2Y731-2 (TQ = 143 s) at 80 K, which has the lowest Y356• yield and therefore the largest contribution of F3Y122•. Experimental parameters: two-pulse echo sequence, /2 pulse length = 12 ns,  = 240 ns, shotrepetition-time = 2 ms, shots-per-point = 150, magnetic field axis resolution = 0.02 mT, 1 scan. A background signal stemming from the resonator and a signal the e' center 13 from the CFQ EPR tubes have been subtracted from the spectrum, contributing less than 10 % to the total signal intensity.

19 F ENDOR at 80 K and comparison to 50 K
Most ENDOR measurements in this work were performed at 50 K to benefit from enhanced electron spin polarization. At 50 K the polarization is increased by 60% compared to 80 K, but the signal of F3Y122• is only suppressed for larger  values (≳ 500 ns). This leads to the requirement of subtracting contributions from F3Y122• in the 19 F Mims ENDOR experiments with smaller  values at T = 50 K. These contributions are suppressed at 80 K. Here, the results of ENDOR measurements at 80 K of two samples are presented and compared to results obtained at 50 K. At both temperatures, identical measurement parameters were used. Figure S13 shows ENDOR spectra of E52QF3Y122-2/F2Y731-2 (TQ = 153 s) at 80 K (black lines) compared to spectra obtained at 50 K (red lines). For the 80 K measurements with short  values (236 and 266 ns, Figure S13A), only 1 H resonances originating from Y356• were subtracted, whereas it was also necessary to subtract 19 F resonances from unreacted F3Y122• in the 50 K measurements. Importantly, the spectra are indistinguishable at the two temperatures, which clearly shows that the background correction procedure to remove 19 F resonances from unreacted F3Y122• could be performed reliably for the measurements at 50 K. Figure S13B shows measurements at 80 K with a larger  value of 620 ns. Again, similar spectra are obtained at both temperatures, albeit the peak intensities are slightly different. This observation was attributed to relaxation effects. Noteworthy, the S/N in the spectra shown in Figure S13B at 80 K is worse by a factor of ~2.5 as compared to 50 K, despite similar measurement times (typically ~20 h per spectrum).

Control experiments with 17 O-Y-2
17 O-Y-2 was expressed to control whether any signs of a short distance between 17 O-Y731() and Y356•() could be obtained via 17 O ENDOR spectroscopy. Aside from the isotopic enrichment of the Y amino acids, 17 O-Y-2 is identical to the wildtype of the  subunit. 17 O-Y-labelled 2 was expressed in analogy to previous protocols 14  The activity of 17 O-Y-2 was assayed in a similar manner as described in section 1. The specific activity of 17 O-Y-2 was found to be identical to the specific activity of wt-2.

Preparation of 17 O-Y-2 and mass spectrometric determination of labelling degree
The labelling degree was confirmed by mass spectrometry (LC-MS/MS) on peptides obtained by digesting 2 protein overnight by either trypsin or chymotrypsin. After C18 clean-up, the resulting peptides were subjected to LC-MS/MS analysis using an UltiMate 3000 RSLCnano HPLC system (Thermo Fisher Scientific) coupled online to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). We used the intensity ratio of the first two isotopic peaks to estimate the 17 O incorporation rate. This way, a labelling degree of 35 -40% was obtained, in agreement with the labelling degree of the 17 O labeled tyrosine as specified by the manufacturer.   17 O ENDOR measurements at 94 GHz/3.4 T were performed using parameters based on our recent 17 O ENDOR publication, 17 but adjustments to increase the measurement sensitivity were attempted (e.g. smaller RF window, longer  values). Three different samples were investigated (Table S2)

DFT Calculations for the simulations of the ENDOR spectra
After extracting the tyrosine triad from the cryo-EM structure, geometry optimization was performed using the unrestricted Kohn-Sham method with the BP86 functional 18,19 and the def2-tzvp basis set. 20 For dispersion correction, Grimme's D3 correction with Becke-Johnson damping was used. [21][22][23] For the optimization, the non-hydrogen and non-fluorine atoms in F2Y731 and Y730 were constrained in their XYZ coordinates. Furthermore, also the O -O distance between Y356• and F2Y731 was constrained. The geometry optimization of the triad aimed at correcting the covalent bond lengths to hydrogen and fluorine as well as the bond lengths in the tyrosyl radical to achieve a reasonable starting structure S1 from which all other models could be derived in the subsequent modelling. These bond lengths could not be obtained correctly from the cryo-EM structure, as it did resolve neither the hydrogen atoms nor the location of tyrosyl radicals and also didn't contain fluorine labels at Y731.
After the described geometry optimization, the HFC parameters were calculated using the B3LYP 19,24 functional with the def2-tzvpp basis set. Additionally, the RIJCOSX approximation was used (auxiliary basis set def2/J). 25,26 An error of ±20 % for the DFT derived EPR parameters is estimated. The CPCM(ethanol) keyword was used to include the assumed polarity of the radical's environment.
As mentioned in the main text, the presence of H-bond donors affects the radical's spin density distribution. H-bond donors reduce the spin density of the O atom of the phenoxyl radical, 27 which affects the coupling constants. In test calculations where we omitted the H-bonding water we could obtain similar coupling strengths as in the presented models S4 and S5 by increasing the O-O distance between F2Y731 and Y356• by 0.1 -0.2 Å.
The calculated HFC parameters were used in the ENDOR simulations with only minor adjustments. First, the ORCA derived Euler angles and HFC constants were rounded to integer numbers (i.e. 1 kHz precision for the HFC constants). When adapting the Euler angles from the ORCA output-files, it was considered that EasySpin uses framerotation, while ORCA uses tensor-rotation. Therefore, the order of the Euler angles in the EasySpin input was reversed with respect to the ORCA output and the sign of each angle was changed, too.
Second, all 19 F HFC tensors were classified as to whether or not they were originating from purely dipolar hyperfine coupling. Purely dipolar interaction requires a vanishing isotropic coupling constant (aiso = 0) and an anisotropic coupling tensor ̿ which is described by Equation (S6) (see also discussion in the main text): Where the dipolar coupling constant T for a 19 F nucleus is given by Equation (S7): R is the distance between the centroid of the O, C1, C3, and C5 atoms of the phenoxyl radical and the 19 F nucleus, accounting for spin delocalization. 28 This dipolar, centerof-gravity approximation breaks down for short distances. Then, no equations like Equations (S6) and (S7) exists. Instead, the more general Equation (S8) has to be used: Nevertheless, the anisotropic coupling will in general increase when the distance between the unpaired electron and the 19 F nucleus decreases.
DFT usually predicts very small deviations from the purely dipolar case (Equations S6 and S7) which cannot be resolved experimentally. 11 Hence, all deviations from the purely dipolar case have been ignored in ENDOR simulations when the calculated isotropic coupling constant aiso amounted to less than 10% of the anisotropic coupling constant T and was below 50 kHz. Likewise, deviations of the DFT derived anisotropic coupling constants from the tensor elements in Equation (S6) have been ignored when they did not exceed 10% of T. In cases of larger deviations, the HFC constants have been used without introducing any approximation (i.e. in accordance with Equation  S8).

Models of Y356•, F2Y731, and Y730
Most models are shown in the main text, here we depict the remaining stacked model S3 in Figure S16 and compare it to S2 and, additionally, show the flipped Y dyad taken from the crystal structure of NH2Y730- (no  subunit) in Figure S17.
To arrive at S3, the phenol plane of F2Y731 in S2 was rotated while monitoring how this affects the coupling of the 19 F nuclei. It was possible to obtain a structure S3 in which couplings of ~250 kHz for the 19 F atom located closer to the radical are predicted by DFT. This value could potentially be assigned to Fb. Furthermore, the second, more weakly coupled 19 F nucleus in S3 has a dipolar coupling constant of ~65 kHz that would fit Fd, but no explanation for the large coupling assigned to Fa is obtained. Figure S16. Comparison of S2 (purple sticks) with S3 (orange sticks). Since only F2Y731 differs markedly between S2 and S3, only this residue is shown for S3. The arrow indicates how S3 was obtained from S2 in PyMOL. Nearby R411 and Y413 (both ) are included as narrow sticks. Figure S17 presents the implementation of the flipped Y-dyad from the crystal structure of NH2Y730-2 14 (Bordeaux) into the cryo-EM structure and compares it to S2 (semitransparent purple). The implementation was achieved by removing the NH2 group from Y730 and adding 1 H and 19 F atoms where needed to the crystallographic dyad. As can be seen, flipped F2Y731 directly derived from the crystal structure clashes with N733 (from the ordered  pair of the cryo-EM structure). Figure S17. Comparison of the crystal structure derived Y-dyad Y730-F2Y731 (Bordeaux) to S2 (semitransparent purple). Three nearby residues of the  subunit taken from the cryo-EM structure are included as thin, blue sticks. A clash with N733 is indicated. Table S6 provides a summary description of each model structure and establishes hierarchical relations between the models. Table S6. Qualitative description of models S1 -S5, including the most significant geometrical modifications leading from one model to the next.

Model Description S1
Y triad from cryo-EM with the addition of F and H atoms where needed S2 Based on S1, Y356• repositioned and realigned by changing dihedral angles around C -Namino, C -C, C -C1,phenol. Centroid of Y356• (based on O, C1, C3, and C5) was moved by ca 1 Å, distance between centroid of Y356• and O of F2Y731 reduced by ca. 0.5 Å.
Key geometrical parameters of the final model structures S1 -S5 are given in Table  S7. The xyz coordinates of each model can be found in Section SI10. We note that many more models were tested, but to keep the amount of text manageable we only include the most significant ones herein.   Table S8 lists all DFT derived 19 F HFC parameters for models S1 -S5. Finally, models S1 -S5 were also used to calculate the expected coupling constants of the phenol O-atom of Y731. These values should allow estimating coupling constants of stacked and flipped conformers in 17 O-Y-2 samples (Table S9).

Optimizing the stacked/flipped ratio in simulations of ENDOR spectra for different samples
The different ENDOR samples (different mutants and quenching times) showed slight variations in their stacked/flipped ratios. The root-mean square deviation (rmsd) between simulated (based on structures S2 and S5) and experimental data was calculated for the sum spectra of both short (236/266 ns) and long (~620 ns)  value measurements in dependence of the ratio of stacked and flipped conformations to find the optimal value. The rmsd was calculated using equation (S9), where I represents the intensity of either simulated or experimental data and N the number of data points: (S9) Figure S18 shows the results of these calculations for each sample in dependence of the percentage of flipped conformer and, equivalently, the ratio stacked/flipped.